Antifungals bk217beta and bk217upsilon and process for producing same

ABSTRACT

THIS DISCLOSURE DESCRIBES TWO NEW ANTIFUNGALS, DESIGNATED BK217B AND BK217$, PRODUCED IN A MICROBIOLOGICAL FERMENTATION UNDER CONTROLLED CONDITIONS USING A NEW STRAIN OF STREPTOVERTICILLIUM CINNAMONEOUS AND MUTANTS THEREOF. THE NEW ANTI-FUNGALS ARE ACTIVE AGAINST A VAREITY OF FUNGI AND THUS ARE USEFUL IN INHIBITING THE GROWTH OF SUCH FUNGI WHEREVER THEY MAY BE FOUND.

y 13, 1971 PING SHU ETAL 3,592,926

ANTIFUNGALS BK217B AND BKZI'IIS AND PROCESS FOR PRODUCING SAME Filed April 14, 1969 2 Sheets-Sheet 1 03 on 8m 8m 8m 0 m m N w m 89 oo co 00 1 00 9 rniuv uzmnawmm 003 com boom 80 Q (.LNHOL-IHd) HONVLiIWSNVHl INVIfN I ORS. PING SHU BY FERDINAND BARBATSCHI ATTORNEY July 13, 1971 PING SHU ETAL ANTIFUNGALS BK217B AND 8X21?!) AND PROCESS FOR PRODUCING SAME Filed April 14, 1969 2 Sheets-Sheet 2 9 m m N w m 03 on com 0% 8m v 0mm r128 0ZmDOwmm oo o ooov o 6 q- N (.LNHQUBd) HQNVLLIWSNVHJ.

INVENTURS. PING SHU FERDINAND BARBATSCHI ATTORNEY United States Patent 3 592,926 ANTIFUNGALS Bimm AND BK217' AND PROCESS FOR PRODUCING SAME Ping Shu, Pomona, N.Y., and Ferdinand Barbatschi,

was isolated from a soil sample collected in Montana. A viable culture of the new microorganism has been deposited with the Culture Collection Laboratory, Northern Utilization Research and Development Division,

5 United States Department of Agriculture, Peoria, 111., fiigf gii g 'af fifif to Amencan Cyanamld Com and has been added to its permanent collection. It is Filed 14 1969 315,806 freely ayallable to the public in this depository under its CL 11; 21 00 accession number NRRL 3594. US. Cl. 424-120 8 Claims The description and identification of this new micro- 10 organism, mainained in the culture collection of the Lederle Laboratories Division, American Cyanamid Com- ABSTRACT OF THE DISCLOSURE pany of Pearl River, N.Y., was supplied by Dr. H. D. This disclosure describes two new antifungals, desig- Tresnrpf these 1abrat?ries' The wi g is a'general Hated BKZHQ and 3142177, produced in a microbio1o description of the organism Streptov ertzczllzum omftam'ogical fermentation under controlled conditions using a NRRL 3594 based on charactenstlcs new Strain of Strepmvem-Cl-llium cinnamoneus and served. The underscored descriptive colors and color chip tants thereof. The new anti-fungals are active against a vadeslgnatlons ff taken from Jacobson Color j riety of fungi and thus are useful in inhibiting the growth mony Mimua 9 Contamer Corporatlon of such fungi wherever they may be found. of Amenca chlcago 111111015- Amount of growth BRIEF SUMMARY OF THE INVENTION Moderate to good on most media; very good on tomato paste oatmeal and potato dextrose agars; light and This invention relates to two new antlfungals, to their restricted on c k l i agan production by fermentation, to methods for their recovery and concentation from crude solutions, and to Aenal mycehum and/Or en masse Spore color processes for their purification. Aerial mycelium whitish; spore masses in pinkish The present invention includes within its scope the shades, ranging from Bisque (4 cc) to Dusty Peach (5 antifungals in dilute forms, as crude concentrates, and ec) to Flesh Pink (5 ca) to Pearl Pink (3 ca) to Nude in pure crystalline forms. These novel products are ac- Ta (4 gc) Sporulation light to heavy depending upon tive against a variety of fungi including Cryptococcus di neoformans and Trichophyton tonsurans. The effects of Soluble pigments the new antifungals on specific microorganisms, together with their chemical and physical properties, differentiate Ye11Wi$h t0 YeHOWISh'bI'OWR Whan P absent on them from previously described antifungals. Several medla- Reverse color DETAILED DESCRIPTION OF THE INVENTION In yellowish to yellowish-brown shades on most medla. The new antifungals which we have designated BK2175 and BK217'y, are formed during the cultivation under 40 Mlsceuaneous physlologlcal reacnons controlled conditions of a new strain of Streptoverticil- Nitrates not reduced; complete liquefaction of gelatin lium cinnamoneus. This new antifungal producing strain in 7 days; melanoid pigment not produced on peptone- TABLE I [Cultural characteristics of Streptoverticillium cinnamoneus NRRL 3594] Incubation: 14 days Temperature: 28 C.

Amount of Soluble Medium growth Aerial mycelium and/or spores pigment Reverse color Remarks Czapeks' solution agar Light, restricted Aerial mycelium whitish, thin. Trace None Pinkish white of pinkish sporulation. Asparagine dextrose agar Moderate do ..do Bamboo (2 fb) Hickey and Tresners agar Good Aerial mycelium whitish, becoming Yellowish; Lt. Amber (3 ic) Bisque (4 ec) to Dusty Peach (5 cc) light. to Cinnamon in sporulation zones. Sporulation (3 le).

eav Yeast extract agar Moderate; restricted... Aerial mycelium whitish becoming None Bamboo (2 lb) to Bisque (4 ec) is sporulation zones. Dk. Luggage Sporulation light. E1 (4 pg). Kusters Oatfiake agar Good Aerial mycelium whitish, becoming Yellowish- Bamboo (2 fb) to Flesh Pink (5 ca) to Bisque (4 ec) in brown; Dk. Luggage Tan sporuiation zones. Sporulation good. light. (4 pg). Tomato paste oatmeal agar. Very good Aerial mycelim whitish, becoming Yellowish- Dk. Luggage Tan Abundant am- Nude Tan. (4 gc) in sporulaing zones. brown; (4 pg) to Ghesther-colored Sporulation good. moderate. nut Brown (4 mi). exudate. Potato dextrose agar Very good Aerial mycelium whitish, becoming Yellowish- Dk. Luggage Tan Bisque (4 ec) to Dusty Peach (5 ec) brown; (4 pg) to Chestin sporulation zones. Sporulation moderate. nut Brown (4 heavy. ni). Bennetts agar Moderate Aerial mycelium whitish, becoming Yellowish- Lt. Amber (3 ic) Bisque (4 co) in sporulating zones. brown; to Dk. Cinna- Sporulation moderate. light. mon (3 1e). Inorganic salts-starch agar d0 Aerial mycelium whitish, becoming None LtTT an (3 gc)..

Pearl Pink (3 ea) in sporulatiou zones. Sporulation moderate.

TABLE II [Mieroinorphology oi Slreptcverticillium cz'mmmoncus NRRL 3504] Aerial mycelium and/or Medium structures sporiicrous Spore shape Spore size Spore surface Inorganic Saltstarch agar branches from long trailing hypae.

Spore chains arising as bivertieillate Spores elliptical (Hi-0.7;; x 1.04.3

Spore surfaces smooth as determined by electron microscopy at 8000);.

to elongate.

TABLE III [Miscellaneous physiological reaction of Sfreptorerticillizmi cimzamoncus NRRL 3594] Temperature: 28 C.

Medium Incubation period Amount of growth Physiological reaction 443% NaCl in yeast extract agar days Nitrates not reduced.

Do. Complete liquefaction.

o. Melanoid pigments not produced.

Tolcrates a maxiuni 0i 7% NaCl in its growth medium.

Micromorphology Spore chains arise as biverticillate branches from long trailing hyphae. Spores elliptical to elongate 0.6-0.7[LX 1.0-1.3,LL. Spore surface smooth as determined by electron microscopy at 8000 The biverticillate nature of the sporophores of NRRL 3594 places it taxonomically in the genus Streptoverticillium. Furthermore, the pinkish spores of the organism relates it to a small group of biverticillate species. When a side by side comparison was made with reference cultures of these organisms, NRRL 3594 was found to correspond most closely in all other essential features to the strains of S. cinnamoneus. Therefore, NRRL 3594 will, hereafter, be considered a strain of that species.

Observations were made of the cultural, physiological and morphological features of the culture in accordance with the methods detailed by Shirling and Gottlieb [Internat. Journ. of Syst. Bacteriol. 162 313-340 (1966)]. Media used in the study were selected from those recommended by Pridham et a1. [Antibiotics Annual 19561957, p. 947- 953] for the cultivation of actinomycetes. Detailed observations are recorded in the previous Tables I, II, III, and 1V below. Underscored descriptive colors are taken from the Color Harmony Manual.

Table 1V.Carbon source utilization pattern of Streptorerticillium cinnamoneus NRRL 3594 Incubation: 14 days Temperature: 28 C.

Carbon source: Utilization 1 Adonitol 0 l-arabinose 0 Galactose 2 d-Fructose 1 i-Inositol 3 Lactose 1 d-lMannitol O d-Melezitose O d-Melibiose 0 d-Raflinose 0 l-rhamnose O Salicin 0 Sucrose 0 d-Trehalose 3 d-Xylose 1 Dextrose 3 Negative control 0 It is to be understood that for the production of the new antifungals the present invention is not limited to this particular organism nor to organisms fully answering the above growth and microscopic characteristics which are given for illustrative purposes. In fact, it is desired and intended to include the use of mutants produced from the described organism by =various means, such as X-radiation, ultraviolet radiation, nitrogen mustard, phage exposure, and the like.

The fermentation process Cultivation of the organism S. cinnamoneus NRRL 3594 may be carried out in a wide variety of liquid culture media. Media which are useful for the production of the novel antibiotic include an assimilable source of carbon such as starch, sugar, molasses, glycerol, etc.; an assimilable source of nitrogen such as protein, protein hydrolysate, polypeptides, amino acids, corn steep liquor, etc; and inorganic anions and cations, such as potassium, sodium, calcuim, sulfate, phosphate, chloride, etc. Trace elements such as boron, molybdenum, copper, etc.; are supplied as impurities of other constituents of the media. Aeration in tanks and bottles by forcing sterile air through or onto the surface of the fermenting medium. Further agitation in tanks is provided by a mechanical impeller. An antifoaming agent such as lard oil may be added as needed.

Inoculum preparation Shaker flask inoculum of Streptoverticillium cinnamoneus NRRL 3594 is prepared by innoculating milliliters of sterile liquid medium in 500 milliliter flasks with scrapings or washings of spores from an agar slant of the culture. The following medium is ordinarily used.

Grams Cerelose 20 Soy flour X200 10 Corn steep liquor 5 Calcium carbonate 3 Water to 1,000 milliliters.

Tank fermentation For the production of the antifungals in tank fermentors the following fermentation medium is preferably used.

Grams Cerelose Soy flour X200 10 Prograsol 5 Sodium chloride 5 Calcium carbonate 1 'Water to 1,000 milliliters.

Prograsol is the trade name for distillers solubles from com Publicker Industries, Philadelphia, Pa. Each tank containing the above sterilized medium is inoculated with 3 to 9% of the inoculum as described above. Aeration is supplied at 0.5 to 1.0 liter of sterile air per liter of mash per minute and the fermenting mixture is agitated :by an impeller driven at 200-800 r.p.m. The temperature is maintained at 24-30 C., usually at 28 C. The fermentation is ordinarily continued for 48 to 200 hours at which time the mash is harvested.

Isolation procedure After the fermentation is completed, the fermented mash containing the antifungals of this invention is filtered, preferably with the addition of diatomaceous earth or any other conventional filter aid. Normally the mycelial cake and filter pad is washed with a small portion of water. Both filtrate and wash are discarded. The mycelial cake is slurry-Washed in chloroform and the antifungals are extracted with methanol using about 1 liter of methanol for every 5 to 7 liters of liquid mash. The methanolic extract is concentrated to an aqueous phase, and a crude solid product containing components BK217u, BK217B and BK217 is formed. The solid product is collected and slurry-washed with acetone and dried over phosphorous pentoxide or other drying agent ordinarily under reduced pressure and at room temperature.

A portion of the above solid is extracted with isopropanol and the extract is evaporated to dryness. The residue is purified by column chromatography on alumina using methanol as eluant using standard techniques and followed by repeated recrystallizations to yield purified BK21704.

To obtain BK217fi and BK217'y another portion of the above solid is extracted with methanol and the methanolic extract is added to approximately two volumes of diethyl ether resulting in the precipitation of crude BK2176. The solid is recovered and dried and then purified first by column chromatography on a Sephadex LH column using 80% methanol in water. The thus-purified BK217B is further purified by use of solvent counter-current distribution following ordinary techniques and using standard two-phase solvent systems. The residue from the above methanolic extract is dried, yielding semi-pure BK217'y. This component also may be further purified by counter-current distribution following ordinary techniques and using standard two-phase solvent systems.

Physical characteristics Carbon 58. 95 60. 22

Hydrogern 8. 17 8. 24

s'a aid 23%? Oxy en sh y None 0. 56

Component BK21713 has no distinct melting point. A standard infrared absorption spectrum of BK217;3 prepared in a KBr pellet is shown in FIG. 1 of the accompanying drawings. This componet exhibits characteristic absorption in the infrared regions of the spectrum at the following wavelengths expressed in microns: 2.97, 3.43, 5.79, 6.12, 6.35, 6.69, 6.92, 7.28, 7.70, 8.63, 9.43, 11.1,11.85, 14.39.

The [3 component in methanol shows ultraviolet absorption maxima at:

The specific rotation of BK2175 is [a] '-=l0.3 (10.4") (c.:0.5 in dimethylformamide).

BK217/3 contains mycosamine, a known and described amino sugar, as part of its molecular structure.

BK2175 gives the following results to selected colortests.

Color responses of BK217B Test: Response Molish Ninhydrin 2,4-dinitrophenylhydrazine Concentrated sulfuric acid Strong violet, brown on standing.

The specific rotation of BK217 is [a] =+231 (i0.5) (c.=0.5 in dimethylformamide.)

BK217 contains mycosamine as part of its molecular structure.

BK2l7'y gives the following results to selected colortests.

Color Responses of BK2l7'y Test: Response Molish Ninhydrin 2,4-dinitrophenylhydrazine Concentrated sulfuric acid Blue, violet on standing.

The novel compounds of the present invention are useful as antifungal agents and possess broad-spectrum antifungal activity in vitro against a variety of standard laboratory microorganisms as determined by the agardilution streak-plate technique. In this assay, the compounds to be tested are made up to contain 2.5 mg. of test compound per milliliter of solution. Observing sterile techniques, two-fold serial dilutions are made of each test solution. One milliliter of each of the original solutions and of each of the serial dilutions is then added to 9 ml. of warm sterile asparagine-meat extract agar capable of supporting growth of the fungal test cultures. The standard sterile nutrient agar solutions containing the different dilutions of the test compounds, along with suitable and comparable control dilutions containing no test compound, are then allowed to cool in Petri dishes thereby forming solidified agar plates. The test yeast-like fungi are prepared for use by growing in broth overnight. The spores of the filamentous fungi are harvested from mature agar slant cultures and are suspended in sterile physiological saline solution. A loopful of each of the resulting live suspensions is then, still employing sterile techniques, streaked upon the surfaces of each of the agar plates and the resulting streaked plates are then incubated. After an appropriate period of time, each of the streaks on each of the plates in inspected visually and the extent, if any, of fungal growth is noted. The minimal inhibitory concentration (expressed in micrograms per milliliter) is defined as the concentration of test compound causing complete inhibition of growth of any particular organism.

In a representative operation, the minimal inhibitory concentration of the compounds of this invention against standard laboratory microorganisms, as determined in the above-described assay, are set forth in Table V below:

The high in vitro antifungal activity of the novel compounds of the present invention makes them useful alone, or in combination with other antifungal agents, to prevent the growth of, or reduce the number of, such organisms as Cryptococcus neoformans and Trichophyton tonsurans present in various environments. They are thus useful in soaps, shampoos and topical compositions for the treatment of wounds and burns. Also, they are useful in wash solutions for sanitation purposes, as in the washing of hands and the cleaning of equipment or furnishings of contaminated rooms or laboratories.

The usefulness of the new antifungals is also demonstrated by their ability to control systemic lethal infections of Cryptococcus neoformans in mice. The test with C. neoformans was run with Carworth Farms CFI female mice, weight about 20 grams, infected intravenously with a lethal dose of a 24 hours trypticase soy broth culture of the organism. The infected mice were treated with BK217/3 or BK217'y in varying dosages by subcutaneous injections within one hour after infection. Tables VI and VII, below, summarize the results obtained with BK2l7 6 and BK2l'7'y against C. neoformans as compared with nystatin, a well-known antifungal. It will be noted that both BK2l7/3 and KB2l7'y are significantly more active than nystatin.

TABLE VI Alive/total mice 3 days after infection BK217B Nystatih Subcutaneous dosage (mg./ kg. of body weight) Norm-Infected, non treated control mice: 1/20.

TABLE VII Alive/total mice 7 days after infection BK217 Nystatin Subcutaneous dosage (mg./ kg. of body weight) N OTE.Infccted, non-treated control mice: /20.

TABLE VIII Alive/total mice 6 days after infection BKZlT/i Nystatin Subcutaneous dosage (mg./ kg. of body weight) N ore-Infected, nontrcated control mice: 0/20.

The invention will be described in greater detail in conjunction with the following specific examples.

EXAMPLE 1 Inoculum preparation A typical medium used to grow the primary inoculum was prepared according to the following formula:

Grams Cerelose 20 Soy flour X200 10 Corn steep liquor 5 Calcium carbonate 3 Water to 1,000 milliliters.

The washed or scraped spores from an agar slant of S. cilznamoneus NRRL 3594 were used to inoculate two, 500 milliliter flasks containing milliliters each of the above sterile medium. The flasks were placed on a rotary shaker and agitated vigorously for 48 hours at 28 C. The resulting flask inoculum was transferred to a 20 liter glass fermentor containing 12 liters of sterile medium. The glass fermentor was aerated with sterile air while growth was carried out for about 48 hours, after which time the contents was used to seed a 300 liter tank fermentor.

EXAMPLE 2 Tank fermentation A fermentation medium is prepared according to the following formula:

Grams Cerelose 1O Soy flour X200 10 Prograsol 5 Sodium chloride 5 Calcium carbonate 1 Water to 1,000 milliliters.

Three hundred liters of the above fermentation medium in a 400 liter stainless steel tank fermentor was sterilized at C. with steam at 15 pounds pressure for 55-60 minutes. The medium was cooled to 28 C. (water cooling jacket), and 12 liters of inoculum, prepared as described in Example 1, was introduced asceptically into the tank. The fermentation was carried out at 28 C., aeration being supplied at the rate of 0.5 liter of sterile air per liter of mash per minute. The mash was agitated by an impeller rotating at a speed of 200 rpm. From time to time when needed, Hodag LG-8 oil Was added as defoaming agent. At the end of approximately 66 hours of fermentation time the mash was harvested.

9 EXAMPLE 3 Isolation of crude BK217u and preparation of crystalline Three hundred liters of fermented mash was filtered with about 2% (w./v.) of diatomaceous earth filter aid and the filter pad washed with about 30 liters of water. The filtrate and wash were both discarded. The mycelial cake was slurry-washed .Wlth about 50 liters of chloroform, the suspension filtered and the chloroform discarded. The Washed cake was extracted by suspending in about 50 liters of methanol with stirring for about onehalf hour. This suspension was filtered and the cake was washed with an additional 20 liters of methanol. The extracts and wash were pooled and the spent mycelial cake was discarded. The combined methanolic extracts were concentrated to an aqueous suspension (volume 4 liters), the solids of which contained a mixture of the three component antifungals; BK217a, BK217B and BK217'y.

The suspension was centrifuged and the solids were slurried in acetone. The supernatant was decanted and the heavy residue (crude BK2l7a) was dried over phosphorous pentoxide under reduced pressure at room temperature, weight 25.3 grams. A portion (20 grams) of the crude BK217a was slurry extracted six times, each with 200 milliliters of isopropanol. The extract was evaporated to dryness and dissolved in about 150 milliliters of ethanol. The insoluble residue was removed by filtration and discarded. 75 milliliters of the ethanolic extract was charged onto an alumina column (60 grams) and the column washed with 240 milliliters of ethanol and then eluted with 500 milliliters of methanol. The progress of the elution was followed by monitoring the optical density of the effluent at 235 and 300 mu. Fractions containing BK217u were collected and concentrated to almost dryness. The solid residue :was dissolved in ethanol and the solution was filtered. The clear filtrate was concentrated to about 4 milliliters. Purified BK217a was precipitated when diethyl ether was added to the ethanolic concentrate. The solid precipitate was collected by centrifugation and washed with diethyl ether. The washed precipitate was dissolved in 5 milliliters of water-saturated butanol and diethyl ether was added until a crystalline solid began to form. The resultant suspension was allowed to stand overnight at 4 C. At the end of the period, the crystalline BK2l7a was separated by filtration, .washed with diethyl ether and dried over P under reduced pressure at room temperature, weight 270 milligrams.

EXAMPLE 4 Isolation of crude BK217,8 and crude BK217 Three hundred liters of fermented mash was filtered with about 2% (w./v.) of diatomaceous filter aid and the filter pad washed with about 30 liters of water. The filtrate and wash were both discarded. The mycelial cake was slurry-washed with about 50 liters of chloroform, the suspension filtered and the chloroform discarded. The washed cake was extracted by suspending in about 50 liters of methanol and stirring for about one-half hour. This suspension was filtered and the cake re-extracted with an additional liters of methanol. The extracts were pooled and the spent mycelial cake was discarded. The combined methanolic extract (volume 78 liters) was concentrated to an aqueous suspension (volume 15 liters) the solids of which contained a mixture of BK21706, BK217B and BK217'y. The suspension was centrifuged and the solids were slurried two times in acetone using 2.0 liters of acetone each time. The remaining wet residue was extracted with two 2.0 liter portions of methanol and the combined methanol extract was added to 8.0 liters of diethyl ether. The precipitate which formed, crude BK217B, was separated by centrifugation and dried over phosphorous pentoxide under reduced pressure at room temperature, weight 21.9 grams. The crude BK217,B .was purified as described in Example 5.

The residue from the above methanolic extraction, crude BK2l7'y, was dried over phosphorous pentoxide under reduced pressure at room temperature, weight 29.1 grams. The thus-obtained crude BK217'y was purified as described in Example 6.

EXAMPLE 5 Preparation of BK217/3 10 grams of crude BK217fi, obtained as described in Example 4, was suspended in about 60 milliliters of dimethylsulfoxide and the suspension extracted with 485 milliliters of methanol in water. The insoluble matter was separated by centrifugation. The clear solution was charged onto a Sephadex LH20 column (lkilogram) [the Sephadex LH20 was prepared for use by slurrying with 80% methanol in Water] and the column was eluted with 80% methanol in water. The progress of the elution was followed by monitoring the optical density of the effluent at 350 and 405 m Fractions containing BK217fl were pooled and concentrated under reduced pressure to an aqueous phase. The precipitate was collected by centrifugation and washed twice with water. About 0.7 gram of BK217,8 was obtained. Fractions containing BK217y, obtained after the elution of BK2l7fi, may be either discarded or saved for later recovery as described in Example 6 (alternative procedure).

A portion (0.56 gram) of BK217B was further purified by the use of solvent counter-current distribution. The solvent system comprised 2 parts methanol, 2 parts chloroform and 1 part 0.1 M sodium acetate buffer at pH 4.6. The counter-current distribution was carried out in a ZOO-tube apparatus using 10 milliliters upper phase and 10 milliliters lower phase per tube. The BK217;8 to be purified was dissolved in 30 milliliters of lower phase and charged into the first three tubes. Two hundred transfers were made. At the completion of the transfers, samples were taken from each tube and the optical density at 350 and 405 m was determined. Appropriate tubes containing BK217 8 were pooled and evaporated to an aqueous phase. The precipitate was separated by centrifugation and washed twice with water. The microcrystalline material thus obtained was dried over P 0 under reduced pressure at room temperature. The yield of pure BK217B was 89 milligrams.

EXAMPLE 6 Preparation of BK217 A portion milligrams) of crude BK2177, obtained as described in Example 4, was purified using countercurrent distribution technique and following essentially the procedure of Example 5. The sample was dissolved in 70 milliliters of lower phase and 35 milliliters of upper phase and charged into the first 7 tubes. The yield of BK217'y was about 20 milligrams.

(Alternative procedure): The BK2l7'y-containing fractions from the Sephadex LH20 column were pooled and evaporated under reduced pressure until a fine crystalline solid began to precipitate. The solid was collected by centrifugation and washed three times with portions of methanol and then with water. After drying over P 0 under reduced pressure at room temperature, 0.11 gram of purified BK217'y was obtained.

What is claimed is:

1. Antifungal BK217/3, a compound which (a) is effective in inhibiting the growth of fungi; and

in its essentially pure crystalline form (b) has an optical rotation [oc] =10.3 (i0.4)

(c.=0.5 in dimethylformamide):

(c) has the following elemental analysis( percent): C,

58.95; H, 8.17; 0, 31.81; N, 1.17; (d) has ultraviolet absorption maxima at:

(e) has a characteristic infrared absorption spectrum as shown in FIG. 1 of the accompanying drawings.

2. A compound as defined in claim 1, antifungal BK217B, in its essentially pure form.

3. Antifungal BK217 a compound which (a) is efiective in inhibiting the growth of fungi; and

in its essentially pure crystalline form (b) has an optical rotation [a] =+23l (i0.5)

(c.=0.5 in dimethylformamide): (c) has the following elemental analysis (percent): C,

60.22; H, 8.24; O, 29.36; N, 1.62;

(d) has ultraviolet absorption maxima at:

(e) has a characteristic infrared absorption spectrum as shown in FIG. 2 of the accompanying drawings.

4. A compound as defined in claim 3, antifungal BK217'y, in its essentially pure form.

5. A process for the production of antifungal BK217,B which comprises cultivating Streptoverticillimn cinnamoneus NRRL 3594 in an aqueous nutrient medium containing assimilable sources of carbohydrate, nitrogen and inorganic salts under submerged aerobic conditions until substantial antifungal activity is imparted to said medium by the production of antifungal BK217/3, as defined in claim 1, and then recovering antifungal BK217B therefrom.

6. A process for the production of antifungal BK217/3 which comprises cultivating Streptoverticillium cinnanzoneus NRRL 3594 in an aqueous nutrient medium containing assimilable sources of carbohydrate, nitrogen and inorganic salts under submerged aerobic conditions 12 for a period of from 48 to 200 hours and at a temperature of from 24 C. to 30 C. until substantial antifungal activity is imparted to said medium by the production of antifungal BK2175, as defined in claim 1, and then recovering antifungal BK217, 3 therefrom.

7. A process for the production of antifungal BKZ17'y which comprises cultivating Streptoverticillium cinnamoneus NRRL 3594 in an aqueous nutrient medium containing assimilable sources of carbohydrate, nitrogen and inorganic salts under submerged aerobic conditions until substantial antifungal activity is imparted to said medium by the production of antifungal BK217'y, as defined in claim 3, and then recovering antifungal BK217'y therefrom. 4

8. A process for the production of antifungal BKZl'ly which comprises cultivating Streptoverticillium cinnamoneus NRRL 3594 in an aqueous nutrient medium containing assimilable sources of carbohydrate, nitrogen and inorganic salts under submerged aerobic conditions for a period of from 48 to 200 hours and at a temperature of from 24 C. to 30 C. until substantial antifungal activity is imparted to said medium by the production of antifungal BK217'y, as defined in claim 3, and then recovering antifungal B2217 therefrom.

References Cited Miller, The Pfizer Handbook of Microbial Metabolites, McGraw-Hill Book Co., Inc., New York, 1961, p. 392. (Nos. 820 and 821).

JEROME D. GOLDBERG, Primary Examiner U.S. Cl. X.R. 19580; 42412l UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent No. Dated July 3.- 97

Invent Ejng Shu, Ferdinand Barbatschi It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:

"mainained" should read maintained recorded in the previous tables" should tables Column 6, line 29, "Figure 8" Column ll, line l, 03 my) should read 303 mu; line 2, "53 r rpa' should read "333 m H";

line l5, maxima at should read maxima at: 345 m, 5 line 16, "=672,) should read =672,)579 m 4--. Column 12, line 25 "B22l7,/" should read BK21T,

Co Lumn 2, line i0, Column 5, line +9, "are read are recorded in should read Figure 2 Signed and sealed this 15th day of February 1972.

(SEAL) Attest:

EDWARD M.FLETCH ER,JR.

ROBERT GOTTSCHALK Attesting Officer Commissioner of Patents FORM POAOSO 110-691 USCOMM-DC 50376-P69 U 5 GOVERNMENT PRINTING OFFICE V989 03B6 334 

